1. CryomiRs: Characterization of a cold-associated family
of microRNAs in E. solidaginis and E. scudderiana
Pierre J. Lyons1, Nicolas Crapoulet2, Kenneth B. Storey3 and Pier Jr Morin1
1Chemistry and Biochemistry Department, Université de Moncton, Moncton, New Brunswick, Canada
2Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada
3Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada
Introduction
Results
Cold-hardy insect larvae, such as the freeze-tolerant Eurosta solidaginis and the freeze-avoiding Epiblema scudderiana, have the ability to endure significant sub-zero temperatures during the winter months. While not unique, this ability is rare and requires exceptional physiological and molecular adaptations. Both overwintering strategies exhibit similarities, such as an entrance into a deep hypometabolic state characterized by substantial modulation of protein translation. Differences do however exist, with E. solidaginis larvae overcoming the stress via extracellular ice formation and E. scudderiana avoiding ice formation altogether by producing high concentrations of carbohydrate cryoprotectants. We hypothesize that microRNAs (miRNAs), small RNA molecules capable of inhibiting mRNA translation, could play a role in the overwintering survival of cold-hardy insect larvae. To test this hypothesis, RNA from cold-stressed and control E. solidaginis and E. scudderiana larvae was isolated and then followed by Ion Torrent high throughput miRNA sequencing. Differential expression of a small sub-set of miRNAs, referred to as “CryomiRs”, was notably observed between the two species suggesting a common signature of miRNAs underlying insect cold adaptation. Validation of this signature in additional natural models of hypometabolism, including mammalian hibernation, is currently underway.
MiRNA expression profiling via Next-Generation Sequencing
Table 1: Mapping statistics of Ion Torrent reads in cold-stressed vs control E. scudderiana larvae.
Figure 1: Length distribution of small RNA reads in control and -15°C-exposed E. scudderiana. The x-axis represents sequence sizes from 16 to 24 nucleotides. The y-axis indicates the read counts for each size.
Figure 2: MiRNA expression in cold-stressed vs. control E. scudderiana larvae. Bars show log2 fold-change in TMM normalized read counts of small RNAs annotated with known D. melanogaster miRNA sequences (miRBase.org). Shown are miRNAs with minimum average read counts of 10 (after TMM normalization) and minimum log2 fold-change of 0.2. (P < 0.05, n = 2). 1
Overview of insect cold hardiness
Oviposition
Gall formation
Overwintering
Metamorphosis
Figure 3: Real-time PCR validation of expression patterns observed for five miRNAs by next-generation sequencing. Relative expression of miR-34-5p, miR-274-5p, miR-275-3p, miR-307a-3p and miR-316-5p quantified by qRT-PCR in control and -15°C-exposed E. scudderiana. Data are normalized transcript levels (mean ± SEM, n=4).
Figure 4: Length distribution of small RNA reads in control and -15°C-exposed E. solidaginis. The x-axis represents sequence sizes from 16 to 24 nucleotides. The y-axis indicates the read counts for each size.
Cold-modulated miRNAs in natural models of hypometabolism
Table 3: A common signature of upregulated and downregulated miRNAs observed in cold-treated E. solidaginis and E. scudderiana via next-generation sequencing.
CryomiRs:
Table 2: MiRNAs that are modulated differently in the freeze-avoiding E. scudderiana and the freeze-tolerant E. solidaginis via next-generation sequencing.
Lyons, et al., 2013
Materials and Methods
Alignment of sequencing data with mature D. melanogaster miRNA sequences from miRBase
cDNA library prep and barcode tagging of RNA samples
Sequence acquisition
Data trimming (removing barcodes, low Q scores, over/undersized sequences)
miRVanaTM miRNA Isolation Kit (small RNA enriched protocol):
2 extractions containing 2 larvae for each condition (+5 °C and -15 °C were used)
Intra-samples variations of read counts were normalized by the Trimmed Mean of M-values (TMM) method
Differential expression was determined by Student’s t-test
Normalisation and Statistical analysis
MiRNA identification
cDNA library preparation and Ion Torrent sequencing
SmallRNA isolation from cold-stressed E. solidaginis and E. scudderiana
Larvae were first incubated at +5 °C (2 weeks) → Sampled
Larvae were then incubated at -5 °C (2 weeks) → Sampled
Larvae were finally incubated at -15 °C (2 weeks) → Sampled
Exposure of E. solidaginis and E. scudderiana larvae to cold stress
Discussion and Conclusion
This project was undertaken to further characterize a miRNA signature solicited at low temperatures in two cold-hardy gall-forming insects. Small enriched RNAs from cold-stressed and control larvae of the freeze-tolerant goldenrod gall fly Eurosta solidaginis as well as the freeze-avoiding goldenrod gall moth Epiblema scudderiana was characterized via Ion Torrent high throughput sequencing. In E. scudderiana, a total of 44 differentially expressed miRNAs were identified between control and cold-exposed larvae with 21 upregulated miRNAs and 23 downregulated miRNAs. Among the most significant changes observed in miRNAs with potential relevance to cold adaptation were elevated miR-1-3p, miR-92b-3p and miR-133-3p levels as well as reduced miR-13a-3p and miR-13b-3p levels in E. scudderiana larvae exposed to cold temperatures. Expression values obtained from next-generation sequencing were also validated by a quantitative PCR approach for five miRNAs; miR-34-5p, miR-274-5p, miR-275-3p, miR-307a-3p and miR-316-5p. Interestingly, a next-generation sequencing-based approach was also undertaken in the freeze-tolerant E. solidaginis and revealed miRNAs that displayed similar expression profiles as the ones observed in cold-exposed E. scudderiana. These include miR-8-5p, miR-10-5p, miR-31a-5p, miR-34-5p, miR-281-3p, miR-316-5p, miR-317-3p, miR-988 (upregulated) and miR-281-3p, miR-307a-5p, miR-965-3p (downregulated). This common signature of cold-modulated miRNAs suggest that a sub-set of miRNAs are differentially expressed in insects during cold adaptation. Investigating this signature in other animal models of cold adaptation, including mammalian hibernators that interestingly also exhibit miR-1 upregulation, will further put the light on cold-responsive miRNAs or CryomiRs.
Acknowledgements
The insect samples were a kind gift of Kenneth B. Storey, PhD, Carleton University, Ottawa. Next-Generation Sequencing was accomplished in collaboration with Nicolas Crapoulet, PhD, of the Atlantic Cancer Research Institute. This project is funded primarily through a NSERC Discovery Grant awarded to PJM and by the New Brunswick Innovation Foundation (NBIF).
Lyons, Poitras, Courteau, Storey and Morin (2012) Identification of differentially modulated microRNAs in cold-hardy insects. CryoLetters. 34:
Lyons, Lang-Ouellette and Morin (2013) CryomiRs: Towards the identification of a cold-regulated family of microRNAs. Comparative Biochemistry and Physiology - Part D, Genomics and Proteomics. 8:
Lyons, Storey and Morin (2015) Expression of miRNAs in response to freezing and anoxia stresses in the freeze tolerant fly Eurosta solidaginis. Cryobiology. 71:
Lyons, Crapoulet, Storey and Morin (2015) Identification and profiling of miRNAs in the freeze-avoiding gall moth Epiblema scudderiana via next-generation sequencing. Molecular and Cellular Biochemistry. In press.
References